In-vitro antiproliferative efficacy of Abrus precatorius seed extracts on cervical carcinoma

Abrus precatorius is a tropical medicinal plant with multiple medicinal benefits whose seeds have not yet been studied against cervical cancer. Herein, we have assessed the antioxidant and antiproliferative properties of seed extracts (ethyl acetate and 70% ethanol) prepared from Soxhlet and Maceration extraction methods against Hep2C and HeLa Cells. We observed that the APE (Sox) extract had a significantly higher total flavonoid content, APA (Mac) extract had a high total phenolic content, and APA (Sox) extract had a high total tannin content. Further, HPLC analysis of extracts revealed the presence of tannic acid and rutin. Moreover, APA (Sox) exhibited the highest free radical scavenging activity. APE (Mac) had the best antiproliferative activity against Hep2C cells, while APA (Sox) had the best antiproliferative activity against HeLa cells. In Hep2C cells, APE (Mac) extract revealed the highest SOD, catalase activity, GSH content, and the lowest MDA content, whereas APA (Mac) extract demonstrated the highest GST activity. In HeLa cells, APA (Sox) extract showed the highest SOD, GST activity, GSH content, and the least MDA content, whereas APA (Mac) extract showed the highest catalase activity. Lastly, docking results suggested maximum binding affinity of tannic acid with HER2 and GCR receptors. This study provides evidence that A. precatorius seed extracts possess promising bioactive compounds with probable anticancer and antioxidant properties against cervical cancer for restricting tumor growth.

Similarly, the phenolic content of A. precatorius seed extracts was determined using a standard calibration curve (y = 0.015x, r 2 = 0.998) of quercetin (20-100 µg/mL) and expressed in mg quercetin equivalents/g of  Total tannins content of A. precatorius seed extracts was estimated using a standard calibration curve (y = 0.0143x, r 2 = 0.998) of tannic acid (20-100 µg/mL) and expressed in mg tannic acid equivalents/g of extract (Fig. 2). The results showed that APA (Sox) seed extract had 98.98 ± 1 mg tannic acid equivalents/g of extract, while APA (Mac) seed extract had 89.41 ± 0.67 mg tannic acid equivalents/g of extract. APE (Sox) and APE (Mac) seed extracts, on the other hand, had less tannic acid equivalents, with 26.15 ± 0.18 mg/g extract and 17.10 ± 0.13 mg/g extract, respectively ( Antioxidant activity. The antioxidant potential of A. precatorius seed extracts were analyzed by DPPH free radical scavenging assay. Quercetin (standard) and the different seed extracts showed variable antioxidant properties. The lower IC 50 value indicates higher radical scavenging activity. The order of radical scavenging activity was APA (Sox) > APA (Mac) > APE (Sox) > APE (Mac). APA (Sox) with an IC 50 value of 14.82 ± 0.85 µg/ mL showed the highest radical scavenging activity among other extracts as shown in Table 1 and Fig. 3 (Graph 4, Supplementary). The standard (quercetin) exhibited an IC 50 value of 5.65 ± 0.29 µg/mL. Antioxidant capacity was also quantified by FRAP assay. FRAP values were obtained using a standard calibration curve (y = 0.001x, r 2 = 0.919) of FeSO 4 (100-1000 µM) and evaluated based on the capacity to reduce ferric (III) iron to ferrous (II) iron. The results were expressed in mM Fe (II) equivalents/g dry weight of seed extract. APA (Mac) had the maximum FRAP value (105,026.4 ± 57.50 mM Fe (II)/g dry weight of seed extract) among other extracts as shown in Table 1. (Graph 5, Supplementary).   www.nature.com/scientificreports/ HPLC analysis. HPLC was performed to measure the polyphenolic flavonoids (rutin), tannins (tannic acid), and alkaloids (piperine) content of A. precatorius seed extracts prepared using different extraction methods. The retention times for the standards (rutin, tannic acid, and piperine) were RT 3.81, RT 3.09, and RT 14.84, respectively. Furthermore, rutin and tannic acid were identified in APA (Sox), APA (Mac), APE (Sox), and APE (Mac) seed extracts and their retention times were similar to the standards (rutin, tannic acid, and piperine). However, piperine was not identified in any of the extracts, as shown in (Fig. 4). The identified compounds and their corresponding quantities are listed in   Antioxidant enzymes activity assay on cells. The antioxidant enzyme activity (SOD, CAT, and GST) as well as non-enzyme content (GSH and MDA) of A. precatorius seed extracts on Hep2C and HeLa cells were measured using IC 50 specific values to estimate the intracellular reduction of reactive oxygen species (ROS). Fig. 6A, the highest SOD enzymatic activity was observed in APE (Mac) extract (6.92 ± 0.24 U/min/mg of protein) as compared to the control cells (2.74 ± 0.02 U/min/mg of protein). SOD activity of APA (Mac) and APA (Sox) was found to be (4.63 ± 0.03 U/min/mg of protein) and 4.07 ± 0.03 U/min/mg of protein, respectively. APE (Sox) has the lowest enzyme activity (3.04 ± 0.02 U/min/mg of protein). Doxorubicin exhibited a significantly higher SOD activity (27.02 ± 0.02 U/min/mg of protein) compared to other compounds such as rutin and tannic acid, which exhibited 13.41 ± 0.05 U/min/mg of protein and 16.05 ± 0.03 U/min/mg of protein, respectively, as given in Fig. 6A. Similarly, in HeLa Cells, APA (Sox) extract had the highest SOD activity (9.68 ± 0.12 U/min/mg protein) compared to APE (Sox) (8.79 ± 0.09 U/min/mg protein). Furthermore, as compared to tannic acid (3.91 ± 0.76 U/min/mg of protein) and rutin (5.07 ± 0.14 U/ min/mg of protein), doxorubicin showed the highest SOD activity (44.67 ± 0.91 U/min/mg of protein) (Fig. 6D).
Glutathione S-transferase activity. The GST activity in the Hep2C cells upon treating with the APA (Mac) extract was observed to be the highest (42.16 ± 0.55 µmoles/min/mg of protein) compared to the control (28.5 ± 0.05 µmoles/min/mg of protein), followed by APA (Mac) and APE (Sox) i.e., 29.37 ± 0.04 µmoles/ min/mg of protein and 38.56 ± 0.06 µmoles/min/mg of protein, respectively. Doxorubicin treatment resulted in maximum GST activity i.e., 60.46 ± 0.02 µmoles/min/mg of protein in comparison to rutin and tannic acid (Fig. 6A). In HeLa cells, treatment with different extracts and standards revealed that APA (Sox) had the highest GST activity (126.35 ± 2.58 µmoles/min/mg of protein), followed by APE (Sox) (105.10 ± 3.29 µmoles/min/mg of protein. Doxorubicin treatment resulted in the highest GST activity (237.35 ± 5.98 µmoles/min/mg of protein) in comparison to rutin and tannic acid as shown in Fig. 6D.
Glutathione content. The non-enzyme content such as glutathione (GSH) was also quantified in the cells in a similar manner as above. Upon quantitative analysis, we found that among all the extracts, APE (Mac) treated Hep2C cells showed maximum glutathione content of 22,435.65 ± 5.76 µg/mg of protein compared to the control (18,363.86 ± 57.90 µg/mg of protein) (Fig. 6B). GSH content in APE (Sox) and APA (Sox) was found  Fig. 6E).
Lipid peroxidation. Malondialdehyde content (MDA) was also estimated and Hep2C cells showed a marked decrease in the MDA content upon exposure to APE (Mac) (0.041 ± 0.89 µg/mg of protein) compared to the control cells i.e., 0.084 ± 0.12 µg/mg of protein (Fig. 6C). APE (Sox) and APA (Sox) had also shown very less MDA content i.e., 0.072 ± 0.08 µg/mg of protein and 0.079 ± 0.01 µg/mg of protein, respectively. On the other hand, in HeLa cells, the MDA content was observed to be minimal when exposed to APA (Sox) i.e.,

Molecular docking analysis.
In the docking analysis, the binding affinities of the identified compounds ( Fig. 7A) were studied against the cervical cancer receptors including Estrogen, Progesterone, Glucocorticoid, VEGF and HER2 (Fig. 7B). The docking analysis revealed that tannic acid shared the maximum binding affinity with HER2 and GCR (− 9.1 kcal/mol and − 9.0 kcal/mol, respectively) as compared to doxorubicin, whereas rutin shared the strongest binding affinity with HER2 (− 8.9 kcal/mol) among other receptors such as estrogen, progesterone and VEGF (Table 4, Fig. 8). The docked complexes were further visualized for their molecular interactions using the Discovery Studio 2021 client as shown in Fig. 8.

Discussion
Cervical cancer remains a burden for women of low-and middle-income countries (LMICs) such as India, South Africa, China and Brazil. In 2018, there were half a million new cases of cervical cancer and 311,365 deaths due to lack of adequate treatment 12 . Currently, the recommended therapeutic regimens include chemotherapy, radiation therapy, and surgery 13 . However, they present several limitations, including side effects or ineffectiveness. Therefore, it is important to search for novel therapeutic agents or drug candidates that are naturally synthesized which will specifically act on the cancer cells without affecting normal cells. Plant extracts and their bioactive compounds play a significant role in prevention of cancer and many more diseases 14 . Plants have proven to be an excellent reservoir of polyphenols, tannins, flavonoids, alkaloids, terpenes, etc. 15 . Recently, more attention has been placed on tannins with the utilization of some herbs such as Phyllanthus emblica, Sanguisorba officinalis, as well as red wine with considerable tannins 16 . Plant-derived chemotherapeutic agents such as cisplatin, carboplatin, paclitaxel, ifosfamide, curcumin, camptothecin, taxol, and combretastatin have been used widely against cervical carcinoma 17 . Keeping all the above points in mind, the present study was designed to evaluate ethyl acetate and 70% ethanol seed extracts of A. precatorius obtained by different extraction methods as a potential therapy against cervical carcinoma by evaluating their antioxidant activity and in-vitro anti-proliferative activity, as well as binding affinity of their polyphenolic flavonoids (rutin) and tannins (tannic acid) against receptors mediating signaling pathways in cervical carcinoma. www.nature.com/scientificreports/ DNA barcoding is a technique that involves isolation of the genomic and/or organelle DNA and sequencing of a conserved region for species identification. It differs from molecular phylogeny in a way that the main goal is not to determine classification but to identify an unknown sample in terms of a known classification 18 . DNA barcode sequences are very short relative to the entire genome and they can be obtained reasonably quickly and cheaply 19 . Here, the plant sample was identified on the basis of DNA barcoding from the Consortium for the Barcode of Life (CBOL) database. Recently, this technique has been widely used for validating plant as well as animal identification. Various known markers for the identification of plants are trnH-PsbA, ITS F, matK, rbcL 20 . Other reports also suggested use of plant barcoding for the identification 19,20 .
Here, we reported that the content of major flavonoids of A. precatorius was significantly higher in APE (Sox) seed extract (112.16 ± 0.9 mg quercetin equivalent/g of extract) as compared to the other extracts. Similarly, the total phenolic content in A. precatorius was higher in APA (Mac) seed extract (108.88 ± 0.4 mg quercetin equivalent/g of extract) in comparison to the other extracts. Moreover, the total tannin content of APA (Sox) amounting to 98.98 ± 1 mg tannic acid equivalents/g of extract was higher than that of other extracts. As corroborated previously, less content of flavonoids, phenolics and tannins of A. precatorius have been reported 23 . Another study reported that polarity of the solvents used for extraction plays an important role in the concentration of phenols and flavonoids [24][25][26] . Indeed, several factors, such as the type of solvent, the extraction process, the part of the plant, temperature, and so on, might influence the yield and extraction of phenolic compounds 27,28 .
It is well established that HPLC can be used to identify, separate and quantify phytochemicals 29 . In our investigation, HPLC analysis aimed at characterization of flavonoid compounds revealed an abundance of rutin in the APA (Sox) (333.44 ± 1.51 mg/g DW) extract whereas tannic acid was observed to be abundant in the APA (Mac) (369.54 ± 1.26 mg/g DW) extract. Literature cites that rutin was less abundant (24.13 ± 1.26 µg/g DW) in   30 . However, to our knowledge, no studies pertaining to the quantification of tannic acid have been reported in A. precatorius, whereas prior studies on the preparation of 80% ethanolic extract of Quercus species has revealed very less tannin content i.e., 127.68 mg/g as compared to the present studies 31 . Antioxidant capacity of the plant extracts mainly depends on both the composition of the extracts as well as the test system. It is influenced by a variety of factors and cannot be evaluated fully using a single approach. To account for the varied mechanisms of antioxidant action, different types of antioxidant capacity measurements should be performed 32 . Hence, we employed different in-vitro assays to get a broader perspective on the antioxidant potential of this plant. APA (Sox) seed extract possesses a significant antioxidant potential as indicated by DPPH free radical scavenging with an IC 50 value of 14.82 ± 0.85 µg/mL as compared to other extracts. Previously, reports have demonstrated that A. precatorius ethanol leaf extracts had antioxidant potential, with IC 50 values of 33.37 μg/mL, 60.67 ± 1.03 µg/mL and 266.13 ± 1.2 µg/mL [32][33][34] . The antioxidant capacity of A. precatorius seed oil has also been demonstrated, with an IC 50 value of 5.03 ± 0.24 mg/mL 35 . The radical scavenging activity can be explained by the different composition of each extract as there are compounds (polyphenolic flavonoids and phenolics) that react quickly with DPPH to get reduced due to the formation of nonradical 36 . As a virtue of their antioxidant properties, polyphenols and tannins may be helpful to human health, as evidenced by past research [37][38][39] .
Total antioxidant capacity was also measured using the FRAP assay, which revealed that the maximum FRAP value in APA (Mac) was 105,026.4 ± 57.50 mM Fe (II)/g DW and 95,601 ± 413.96 mM Fe (II)/g DW in APA (Sox), whereas previous studies found 8.91 ± 0.31 mg TEAC/g DW and 3.69 ± 0.13 mg AAE/g DW in ethanol seed extract, respectively 23 . This suggests that APA (Mac) has a strong ability to react with free radicals in order to change them into more stable non-reactive species and stop radical chain reactions.
We have reported for the first time the potential anticancer activity of A. precatorius seed extracts on Hep2C and HeLa cells as well as their interaction with cervical cancer receptors. All seed extracts exhibited antiproliferative activity in a dose-dependent manner. However, our results revealed that A. precatorius APE (Mac) was the www.nature.com/scientificreports/ most promising extract, with an IC 50 value of 85.91 ± 6.7 µg/mL in the Hep2C cells, among others. Prior studies on Hep2C cells suggests that vulpinic acid, a major key phytocompound, has an IC 50 value of 34.4 µM strongly suppresses cancer cell proliferation and acts as an anticancer agent through an underlying apoptotic mechanism 40 . Similarly, in HeLa cells, APA (Sox) with IC 50 of 26.26 ± 1.09 µg/mL showed highly effective inhibitory activity as compared to the other extracts. Moreover, previous studies on HeLa Cells reported that ethyl acetate extract of A. precatorius roots had anticancer activity with an IC 50 of 11.89 ± 0.63 µg/mL 41 . Since rutin and tannic acid were identified in extracts prepared by various methods, these were independently screened for antiproliferative activity using Hep2C and HeLa cells. In our study, we revealed that tannic acid had the maximum inhibitory activity against Hep2C and HeLa cell lines, whereas prior research had reported no data evaluating antiproliferative efficacies on Hep2C and HeLa cells, but had observed suppression of other cell lines 16 . Previous studies on rutin have shown the anticancer potential for a human renal cancer cell line (786-O) with an IC 50 of 50 µM 42 . This implies that polyphenolic flavonoids and tannins have anticancer properties through suppression of multiple oncogenic signalling pathways and tumor-promoting mechanisms 16 . However, to understand the exact mechanism of specificity against cervical cancer cells, further in-depth and extensive investigations are required.
Antioxidant enzymes such as GST, CAT and SOD catalyze the intracellular reduction of reactive oxygen species (ROS). Nevertheless, sometimes, antioxidant defence mechanisms are not sufficient to maintain a redox balance that promotes prooxidants. Oxidative stress occurs in the body, resulting in toxicity and genetic damage 43 .
The enzymatic activity and non-enzyme content were determined in Hep2C and HeLa cells. Our results revealed that in Hep2C cells, APE (Mac) extract revealed the highest SOD, catalase activity, GSH content, and the lowest MDA content, whereas APA (Mac) extract demonstrated the highest GST activity. Similarly, in HeLa cells, APA (Sox) extract showed the highest SOD, GST activity, GSH content, and the least MDA content, whereas APA (Mac) extract showed the highest catalase activity. Previous studies on extracts of Ocimum sanctum defatted seeds possessing polyphenolic flavonoids demonstrated its antioxidant enzyme activity in-vitro on Rat PC-12 cells 44 . However, no previous research on the antioxidant enzyme activity of A. precatorius seed extracts on cervical cancer cells has been done, to our knowledge. The effects of A. precatorius seed extracts on oxidative stress biomarkers could indicate that targeted interaction with the complex chain of cellular redox processes, such as increased antioxidant enzyme activity and decreased MDA levels in cervical cancer cells, can lead to an imbalance in antioxidant defence mechanisms, with a tendency toward pro-oxidants, a substrate for the cytotoxic effect.
To infer the role of the identified compounds and their association with cervical cancer receptors, we followed an in-silico pipeline. For this analysis, we studied crucial receptors like Glucocorticoids, HER2, VEGF and Hormonal Receptors (ER & PR) known to be associated with proliferation of cervical cancer 4-6 . Our insilico investigations revealed that tannic acid showed the maximum binding energy against HER2 receptor and GCR compared to the standard (doxorubicin). Moreover, the literature cites the association of other important compounds like tangeretin, wogonin, quercetin, and other flavonoids that have shown lesser binding affinities with GCR and HER2 45,46 .

Conclusion
The present study concludes that both the extraction methods (Maceration & Soxhlet) were effective in obtaining a maximum amount of biologically active compounds. We are among few studies that have looked at the anticancer activity of A. precatorius seed extracts on Hep2C and HeLa cells. This information is supported by HPLC quantification of rutin and tannic acid, which can be exploited to isolate bioactive components from A. precatorius seeds in the future. To the best of our knowledge, this is among the first reports to focus on the enzymatic and non-enzyme content in A. precatorius derived seed extracts.
Herein, we have also highlighted the association of a few identified compounds with crucial cervical cancer receptors, which had not been investigated earlier. Therefore, in line with the observations from this pilot study, our ongoing investigations are based on synthesizing of metal nanoparticles and checking their anticancer efficacy in cervical cancer cell lines. This approach is aimed at developing novel therapeutic strategies for cervical cancer management.
In summary, this plant possesses promising compounds to be tested as potential anticancer and antioxidant candidates for treatment of cervical cancer. However, further investigations need to be undertaken either to isolate the anticancer compounds or to determine the in-vivo biological activity of these extracts in order to promote them as potential cervical cancer models for preclinical trials.

Methods
Chemicals. The analytical grade chemicals were purchased from Hi-Media and Merck, India. Standard drugs were purchased from Sigma-Aldrich, India. 2,4,6-tris(2-pyridyl)-s-triazine TPTZ and MTT reagent were procured from Merck, India. Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS) and penicillin (5000 U/mL), streptomycin (2500 U/mL) were purchased from Gibco (USA). Genomic DNA isolation and molecular identification. Genomic DNA was isolated from seeds of A. precatorius using Cetyltrimethylammonium bromide (CTAB) method 47  www.nature.com/scientificreports/ The genomic DNA was amplified using rbcLa-forward primer (ATG TCA CCA CAA ACA GAG ACT AAA GC) and rbcLa-reverse primer (GTA AAA TCA AGT CCA CCR CG). PCR amplification was carried out in Veriti model of Applied Biosystem Thermo cycler 48 . The amplified product was then run on agarose gel and the appropriate band was sliced from the gel and was further processed for DNA elution using Gel Purification kit (Qiagen). The purified product was then utilized for sequencing using universal primers.

Sample collection. Seeds of
Human cervical cell line culture. Human cervical cancer cell lines Hep2C and HeLa were obtained from National Centre for Cell Sciences (NCCS), Pune, India. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) FBS, 100 U/ml penicillin, and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO 2 .
Preparation of extracts. Seeds were washed with distilled water to remove dirt and soil particles, followed by drying and grinding to form powder and used throughout the study.
Soxhlet extraction method. Ethyl acetate and 70% ethanol were used to prepare A.precatorius seed extracts. 10 g of seed powder was placed inside a thimble made from thick filter paper and loaded into the soxhlet extractor. The soxhlet extractor was placed onto the flask containing the solvent (500 mL) equipped with a condenser. The extractor was then allowed to heat to reflux for 16 h at 70 °C. Extracts were filtered twice through a Whatman No.1 paper filter and concentrated to the dry mass with the aid of rotary evaporator 49 . Maceration extraction method. 10 g of seed powder was soaked in 100 ml of solvent (Ethyl acetate and 70% Ethanol) and stored at room temperature for 7 days. The conical flasks of the extract were covered with cotton plugs to avoid the evaporation. After 7 days of incubation, they were filtered with muslin cloth followed by Whatman No.1 filter paper and concentrated to the dry mass with the aid of rotary evaporator 50 .
The dried extracts were dissolved in absolute dimethyl sulfoxide (DMSO) as 50 mg/mL and diluted with phosphate-buffered saline (PBS, pH 7.4) to give final concentrations.
Quantification of phytochemical constituents. The total flavonoid content was measured by the Aluminium Chloride Spectrophotometric method. Absorbance was measured against the prepared blank at 510 nm and results were represented as quercetin equivalents (mg QE)/g of extract. Similarly, total phenolic content and total tannins content were quantified by the Folin-Ciocalteau method. Absorbance of the mixture was measured at 725 nm. Final results were represented as quercetin equivalents (mg QE)/g of extract and tannic acid equivalents (mg TA)/g of extract, respectively 44,49,51 . All the concentrations were calculated using a standard calibration plot.
Antioxidant activity. Antioxidant potential in the seed extracts was determined by electron transfer assay i.e. (2,2-Diphenyl-1-picrylhydrazyl) Radical scavenging assay (DPPH) and Ferric reducing antioxidant power (FRAP) assay. DPPH free radical scavenging assay was performed to measure the hydrogen donating or radical scavenging ability in a dose dependent manner at concentration (1-21 µg/mL) of quercetin and APA (Sox), (1-29 µg/mL) of APA (Mac), (150-170 µg/mL) of APE (Sox) and (150-650 µg/mL) of APE (Mac). Briefly, a 0.04 mM DPPH radical solution was prepared in methanol and then 900 μL of this solution was mixed with 100 μL of extract solution containing different concentrations of seed extracts. The absorbance was measured at 517 nm after 30 min of incubation. Methanol (95%) and DPPH solution were used as blank and control, respectively. Quercetin was used as the standard. 50% inhibitory concentrations (IC 50 values) of the extracts were calculated from a graph as concentration versus percentage inhibition. Radical scavenging activity was expressed as the percentage of inhibition. Measurements were taken in triplicate. The IC 50 of the extract and standards were determined graphically 52 .
The percentage of inhibition was calculated by using the formula: Further, for the FRAP assay, FRAP reagent solution was prepared with 300 mM sodium acetate buffer (pH 3.6), 10 mM 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) in 40 mM HCl and 20 mM FeCl 3 ·6H 2 O (10:1:1 v/v/v). The absorbance was measured at 593 nm after a 30 min incubation at room temperature against 50% ethanol as blank. A calibration curve was prepared using FeSO4 × 7H2O. FRAP values were expressed as mM Fe (II)/g dry weight of extract 49,53,54 . High pressure liquid chromatography. The presence of phenolic compounds and alkaloids in the prepared extracts was screened against standards (rutin, tannic acid and piperine) by HPLC. The analysis was performed using a C-18 reversed phase column (Phenomenex, Gemini 5 μ, 250 mm length × 4.6 mm internal diameter). The mobile phase consisted of methanol: 0.1% orthophosphoric acid (77:23) for rutin, methanol: water (50:50) for tannic acid and 1% acetic Acid: acetonitrile (52:48) for piperine were chosen for the separation at a constant flow rate of 1 mL/min. The column temperature was set to 38 °C and the injection volume was 2 μL. The wavelengths were set to 370 nm for the detection of rutin, 280 nm for tannic acid, and 343 nm for piperine. To plot the standard calibration curves, standard stock solutions of rutin, tannic acid, and piperine were www.nature.com/scientificreports/ produced in methanol at various concentrations (5-100 µg/mL). The results were expressed as milligrams of each compound per g of dry weight (DW) of the extract 49 .
The percent inhibition was calculated by using the following formula: Antioxidant enzyme activity assay on Hep2C and HeLa cells. To  Statistical analysis. The statistical data were represented as mean ± standard deviation (SD) from three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparisons test. A probability value of ≤ 0.05 was considered as statistically significant. All analyses were performed using GraphPad Prism version 8.0.2.